**2. General principles of MFCs and various microbial catalysts**

#### **2.1 Mechanism of electron generation in microbial cells**

MFCs utilize the decomposition energy of organic matters by the organisms to produce ATP, known as the energy currency, based on the energy obtained from

**53**

**Figure 3.**

*Catalyst Development of Microbial Fuel Cells for Renewable-Energy Production*

this process. Taking glucose decomposition in *Saccharomyces cerevisiae* as an example (**Figure 3**), glucose taken into the microbial cells by cell membrane enzymes is oxidized and decomposed to pyruvic acid by various enzymes via an intracellular glycolysis system. Next, pyruvic acid becomes carbon dioxide and water when it is completely oxidized via the TCA cycle. The electrons generated are then collected in the mitochondrial inner membrane in eukaryotes, and in prokaryotes, they were accumulated in the cell membrane via NADH and FADH2 (both of which provide two electrons to the membrane electron flow; **Figure 3**). In addition, the flow of electrons in these membranes is maintained through multiple protein complexes localized in the membrane. Quinone compounds and cytochrome proteins are also included in the complex. ATP is synthesized by the membrane enzyme, ATP synthase, using the proton concentration gradient, which is generated inside and outside the membrane because of the flow of electrons. In MFCs, it is thought that it takes the electrons directly from NADH or the flow of electrons generated in the membrane by the decomposition of organic substances. A part of the electrons generated within the microbial cell is carried to an electrode outside the cell via an electron carrier called a mediator. When one molecule of glucose is completely oxidized into carbon dioxide and water in the cell, 10 molecules of NADH and 2 molecules of FADH2 are also generated. In total, 24 electrons are obtained from 1 glucose molecule. If this principle were to apply to fuel cells, the Coulomb efficiency, which is an index of energy efficiency, would become 100%. Therefore, in order to obtain electrons more efficiently from the cells via such metabolism, it is important to adjust the redox balance within the microbial cells in the MFCs.

In the case of using glucose as the fuel source, the reaction occurring in the anode tank is represented by Eq. (1), and the reduction reaction occurring in the

<sup>−</sup> (1)

**2.2 Calculation of the energy obtained from glucose**

C6H12O6 + 6H2O → 6CO2 + 24H<sup>+</sup> + 24e

cathode tank is represented by Eq. (2).

*DOI: http://dx.doi.org/10.5772/intechopen.81442*

*Glucose metabolism (Saccharomyces cerevisiae).*

*Catalyst Development of Microbial Fuel Cells for Renewable-Energy Production DOI: http://dx.doi.org/10.5772/intechopen.81442*

#### **Figure 3.**

*Current Topics in Biochemical Engineering*

the level of several W/m3

[16]. Other fuel cells performing beyond 500 W/m3

The performance of MFCs is evaluated based on some indicators. The electrical energy (Wh) used to express the capacity of dry batteries is also an important indicator, but only a few papers have reported it so far. In most cases, the performance is expressed as the maximum power per anode electrode area (power density per area) or the maximum power per cell volume (power density per volume). The latter is a straightforward index and important for practical use. For example, a relatively high-performance small-scale fuel cell (2.5 mL) using a complex (mixed) microorganism system was reported in 2007 with a power density of 1550 W/m3

many of them are still at a microliter or milliliter scale. Owing to the low proton diffusion speed and high internal resistance, the maximum power per volume tends to be small for a large-scale fuel cell. MFCs with a volume more than 1 L were also being studied in the laboratory, but the maximum power per volume was still low at

Practical applications of MFCs are still problematic because of the high cost and low-power generation. Despite this situation, research on the implementation of MFCs has been carried out. For example, an artificial stomach called Gastrobot (aka Chew-Chew train) using *E. coli* and sugar as the catalyst and fuel, respectively, EcoBot-III (a self-sustainable robot with its own circulatory system, such as ingestion, digestion, and excretion), and several environmental sensors using the MFCs for powering [2]. Such implementation studies are also important to understand the desired performance level for MFCs. Meanwhile, in order to bring MFCs closer to practical use, a combination of power generation and other effects is one of the promising methods. For example, MFCs that are installed at a wastewater treatment facility are expected not only to reduce the generated sludge amount from the treatment but also to cover a part of the electricity load used by the plant. Recently, the minimum performance of MFCs required for reaching energy neutrality in a wastewater treatment facility has been calculated [25], and the realization has been highly expected. Such research on MFCs installed for wastewater treatment has been actively carried out so far, and the experiments were examined at a pilot-scale plant of more than 10 L [26–28]. Accordingly, the practical use of MFCs is expected soon. Besides this, although it deviates from energy production, the use of MFCs as a sensor has also been extensively studied. In order to perform on-site real-time monitoring, it is important to recognize the toxic compounds rapidly. Several analytical techniques based on electrochemistry have been developed for this purpose, but in many cases, they lack practicality for environmental measurements. The MFC-based biosensor is one promising candidate, and it has already been shown that not only toxic heavy metals but also toxic organic compounds can be detected [29]. The sensitivity adjustment suitable for the detection of specific contaminants

is left as a challenge, but there is still a great expectation for its application.

**2. General principles of MFCs and various microbial catalysts**

**2.1 Mechanism of electron generation in microbial cells**

Here, we will explain the mechanism of electron generation in microorganisms, introduce the principle of MFCs, describe the microbial catalysts used for various MFCs mentioned above, and discuss the recent topics on microbial catalysts.

MFCs utilize the decomposition energy of organic matters by the organisms to produce ATP, known as the energy currency, based on the energy obtained from

to tens of W/m3

MFCs, and further improvements are still being conducted.

were also reported [17–22], but

[23, 24]. Scaling-up is also another issue of

**52**

*Glucose metabolism (Saccharomyces cerevisiae).*

this process. Taking glucose decomposition in *Saccharomyces cerevisiae* as an example (**Figure 3**), glucose taken into the microbial cells by cell membrane enzymes is oxidized and decomposed to pyruvic acid by various enzymes via an intracellular glycolysis system. Next, pyruvic acid becomes carbon dioxide and water when it is completely oxidized via the TCA cycle. The electrons generated are then collected in the mitochondrial inner membrane in eukaryotes, and in prokaryotes, they were accumulated in the cell membrane via NADH and FADH2 (both of which provide two electrons to the membrane electron flow; **Figure 3**). In addition, the flow of electrons in these membranes is maintained through multiple protein complexes localized in the membrane. Quinone compounds and cytochrome proteins are also included in the complex. ATP is synthesized by the membrane enzyme, ATP synthase, using the proton concentration gradient, which is generated inside and outside the membrane because of the flow of electrons. In MFCs, it is thought that it takes the electrons directly from NADH or the flow of electrons generated in the membrane by the decomposition of organic substances. A part of the electrons generated within the microbial cell is carried to an electrode outside the cell via an electron carrier called a mediator. When one molecule of glucose is completely oxidized into carbon dioxide and water in the cell, 10 molecules of NADH and 2 molecules of FADH2 are also generated. In total, 24 electrons are obtained from 1 glucose molecule. If this principle were to apply to fuel cells, the Coulomb efficiency, which is an index of energy efficiency, would become 100%. Therefore, in order to obtain electrons more efficiently from the cells via such metabolism, it is important to adjust the redox balance within the microbial cells in the MFCs.

#### **2.2 Calculation of the energy obtained from glucose**

In the case of using glucose as the fuel source, the reaction occurring in the anode tank is represented by Eq. (1), and the reduction reaction occurring in the cathode tank is represented by Eq. (2).

$$\text{C}\_6\text{H}\_{12}\text{O}\_6 \star \text{6H}\_2\text{O} \rightarrow \text{ 6CO}\_2 + 24\text{H}^+ + 24\text{e}^- \tag{1}$$

$$\text{6O}\_2 + 24\text{H}^+ + 24\text{e}^- \rightarrow \text{ 12H}\_2\text{O}.\tag{2}$$

The oxidation-reduction potential of Eq. (1) is −0.42 V, whereas the oxidation-reduction potential of Eq. (2) is 0.82 V. Therefore, the total potential difference of the MFC reaction (Eq. (1) + Eq. (2)) as represented by Eq. (3) is 1.24 V. Theoretically, the voltage exceeds 1 V, but in most cases, it has never reached that value.

$$\text{C}\_6\text{H}\_{12}\text{O}\_6 \star \text{6O}\_2 \star \text{6H}\_2\text{O} \rightarrow \text{ 6CO}\_2 + 12\text{H}\_2\text{O}.\tag{3}$$

Assuming that 24 electrons are obtained from 1 glucose molecule and that they can be recovered in 1 h, the quantity of electricity (Ah) obtained from the glucose (1 kg) can be calculated using the Faraday constant (96,485 C/mol) as shown in Eq. 4. As a result, the electrical energy of 4430 Wh can be achieved if the potential is 1.24 V; accordingly, this value is the same as the value mentioned in the Introduction section.

$$(24 \times 96, 485 \times 1000) / (3600 \times 180) \, = \, 3574 \,\text{Ah}.\tag{4}$$

#### **2.3 Basic components of dual-chambered MFCs using a mediator**

A dual-chambered fuel cell consisting of an anode tank and a cathode tank is the simplest and has been used for a long time for MFCs. In many cases, they are separated by a cation exchange membrane (CEM) to create a potential difference between the two tanks (**Figure 4**). CEM prevents mixing of each content and allows the protons generated in the anode to migrate to the cathode. In addition, CEM selection, especially based on its proton transfer efficiency, is important

**55**

*Catalyst Development of Microbial Fuel Cells for Renewable-Energy Production*

because it significantly regulates the movement of the protons responsible for the pH reduction at the anode affecting the activity of microorganisms and the delivery of electrons to the oxygen at the cathode. Also, some factors to consider, such as durability and cost, are important for selecting CEM. At present, Nafion is popular

Numerous research studies are being conducted to evaluate the influence of the electrode materials on the performance and cost of the MFCs. Carbon materials, which are noncorrosive, have been widely used because of their high electrical conductivity and chemical stability, e.g., carbon rod, carbon fiber, carbon felt, and carbon cloth [3]. Biocompatibility, specific surface area, electrical conductivity, and cost are important factors for its selection. Since its discovery in 2004, graphene has been attracting much attention for its use as an electrode because of its high specific surface area, electrical conductivity, and biocompatibility [32]. In fact, graphene has been already used in lithium-ion batteries, and the development of graphenemodified materials to increase the power density has progressed actively [33, 34]. Moreover, since biofilm formation by microorganisms on the electrodes affects the performance of MFCs, the preference of electrode materials tends to shift from two-dimensional to three-dimensional surfaces, where a larger surface area is obtained; thus, the contact with microorganisms increases. Furthermore, metals are also used as the electrodes. The conductivities are higher than those of carbon materials, but they are prone to corrosion in the anode solution. Therefore, metals are problematic to use, except for stainless and titanium. To improve such problem,

materials in which metal is incorporated into graphite have been made [3].

A phosphate buffer or bicarbonate buffer solution is often used for the anode electrode solution to achieve high performance [16, 35]. The pH of the solution affects not only the activity of microorganisms but also the transfer of hydrogen ions used from the anode to the cathode when the electrons are transferred to oxygen at the cathode. The solutions contain microorganisms as the catalyst, organic matter as the fuel, and mediator as the electron carrier. In addition, there are reports that the performance of MFCs was improved by adding NaCl to increase

Regarding the fuel, many substrates have already been studied [37]. For example, acetic acid, lactic acid, glycerol, glucose, xylose, sucrose, starch, yeast extract, malt extract, various real wastewaters, and synthetic wastewater were used depending on the purpose of each research. Generally, the fermentable substrate of microorganisms is used to generate electricity more efficiently. There is a trend where glucose is used when using *S. cerevisiae*, lactic acid when using *S. oneidensis*, and acetate when using *G. sulfurreducens* in the experiments. On the basis of a calculation, when lactic acid, acetic acid, and glycerol are completely oxidized, there are 14, 8, and 14 electrons obtained, respectively. The number of electrons obtained

Regarding the mediator, although some microorganisms can carry electrons directly to the electrode as described later, in many cases, the electrons cannot be carried, or the performance is low even if carried, so an artificial mediator that can pass through the cell membrane is added to the anode solution. The typical compounds for artificial mediators are methylene blue, neutral red, 2-hydroxy-1,4-naphthoquinone (HNQ ), thionine, benzyl viologen, 2,6-dichlorophenolindophenol, and various phenazines [38]. It was reported that the hydrogenase donates electrons to the neutral red [39], but the process was not yet clearly proven as to how these mediators deprive electrons of the cell. It is thought that, depending on the type of mediator, the electrons may be taken directly from NADH or obtained from the electron transfer system of the cell membrane. On the other hand, there is also a difficult aspect of using a mediator. In order to increase the electron transfer

from each substrate depends on the metabolic pathway.

*DOI: http://dx.doi.org/10.5772/intechopen.81442*

for many MFCs [30, 31].

the ionic strength [36].

**Figure 4.**

*General dual-type MFC. Med: mediator, CEM: cation exchange membrane.*

#### *Catalyst Development of Microbial Fuel Cells for Renewable-Energy Production DOI: http://dx.doi.org/10.5772/intechopen.81442*

because it significantly regulates the movement of the protons responsible for the pH reduction at the anode affecting the activity of microorganisms and the delivery of electrons to the oxygen at the cathode. Also, some factors to consider, such as durability and cost, are important for selecting CEM. At present, Nafion is popular for many MFCs [30, 31].

Numerous research studies are being conducted to evaluate the influence of the electrode materials on the performance and cost of the MFCs. Carbon materials, which are noncorrosive, have been widely used because of their high electrical conductivity and chemical stability, e.g., carbon rod, carbon fiber, carbon felt, and carbon cloth [3]. Biocompatibility, specific surface area, electrical conductivity, and cost are important factors for its selection. Since its discovery in 2004, graphene has been attracting much attention for its use as an electrode because of its high specific surface area, electrical conductivity, and biocompatibility [32]. In fact, graphene has been already used in lithium-ion batteries, and the development of graphenemodified materials to increase the power density has progressed actively [33, 34]. Moreover, since biofilm formation by microorganisms on the electrodes affects the performance of MFCs, the preference of electrode materials tends to shift from two-dimensional to three-dimensional surfaces, where a larger surface area is obtained; thus, the contact with microorganisms increases. Furthermore, metals are also used as the electrodes. The conductivities are higher than those of carbon materials, but they are prone to corrosion in the anode solution. Therefore, metals are problematic to use, except for stainless and titanium. To improve such problem, materials in which metal is incorporated into graphite have been made [3].

A phosphate buffer or bicarbonate buffer solution is often used for the anode electrode solution to achieve high performance [16, 35]. The pH of the solution affects not only the activity of microorganisms but also the transfer of hydrogen ions used from the anode to the cathode when the electrons are transferred to oxygen at the cathode. The solutions contain microorganisms as the catalyst, organic matter as the fuel, and mediator as the electron carrier. In addition, there are reports that the performance of MFCs was improved by adding NaCl to increase the ionic strength [36].

Regarding the fuel, many substrates have already been studied [37]. For example, acetic acid, lactic acid, glycerol, glucose, xylose, sucrose, starch, yeast extract, malt extract, various real wastewaters, and synthetic wastewater were used depending on the purpose of each research. Generally, the fermentable substrate of microorganisms is used to generate electricity more efficiently. There is a trend where glucose is used when using *S. cerevisiae*, lactic acid when using *S. oneidensis*, and acetate when using *G. sulfurreducens* in the experiments. On the basis of a calculation, when lactic acid, acetic acid, and glycerol are completely oxidized, there are 14, 8, and 14 electrons obtained, respectively. The number of electrons obtained from each substrate depends on the metabolic pathway.

Regarding the mediator, although some microorganisms can carry electrons directly to the electrode as described later, in many cases, the electrons cannot be carried, or the performance is low even if carried, so an artificial mediator that can pass through the cell membrane is added to the anode solution. The typical compounds for artificial mediators are methylene blue, neutral red, 2-hydroxy-1,4-naphthoquinone (HNQ ), thionine, benzyl viologen, 2,6-dichlorophenolindophenol, and various phenazines [38]. It was reported that the hydrogenase donates electrons to the neutral red [39], but the process was not yet clearly proven as to how these mediators deprive electrons of the cell. It is thought that, depending on the type of mediator, the electrons may be taken directly from NADH or obtained from the electron transfer system of the cell membrane. On the other hand, there is also a difficult aspect of using a mediator. In order to increase the electron transfer

*Current Topics in Biochemical Engineering*

that value.

Introduction section.

6O2 + 24H<sup>+</sup> + 24e

The oxidation-reduction potential of Eq. (1) is −0.42 V, whereas the oxidation-reduction potential of Eq. (2) is 0.82 V. Therefore, the total potential difference of the MFC reaction (Eq. (1) + Eq. (2)) as represented by Eq. (3) is 1.24 V. Theoretically, the voltage exceeds 1 V, but in most cases, it has never reached

C6H12O6 + 6O2 + 6H2O → 6CO2 + 12H2O. (3)

(24 × 96,485 × 1000)/(3600 × 180) = 3574 Ah. (4)

A dual-chambered fuel cell consisting of an anode tank and a cathode tank is the simplest and has been used for a long time for MFCs. In many cases, they are separated by a cation exchange membrane (CEM) to create a potential difference between the two tanks (**Figure 4**). CEM prevents mixing of each content and allows the protons generated in the anode to migrate to the cathode. In addition, CEM selection, especially based on its proton transfer efficiency, is important

**2.3 Basic components of dual-chambered MFCs using a mediator**

*General dual-type MFC. Med: mediator, CEM: cation exchange membrane.*

Assuming that 24 electrons are obtained from 1 glucose molecule and that they can be recovered in 1 h, the quantity of electricity (Ah) obtained from the glucose (1 kg) can be calculated using the Faraday constant (96,485 C/mol) as shown in Eq. 4. As a result, the electrical energy of 4430 Wh can be achieved if the potential is 1.24 V; accordingly, this value is the same as the value mentioned in the

<sup>−</sup> → 12H2O. (2)

**54**

**Figure 4.**

efficiency by the mediator, it requires a high concentration, but because of its high toxicity, it has a strong influence on the cells; therefore, the level of use is necessary to be controlled.

Finally, the cathode solution is explained as follows. The electrons generated at the anode are carried to the cathode, where the reduction reaction takes place. When oxygen, the most common electron acceptor, is used as an oxidizing agent, aeration is necessary because oxygen has low solubility (about 8 mg/L DO). There are cases where oxygen generation by the photosynthesis of algae is used for oxygen supply [40, 41]. In the reaction at the cathode, H2O is produced by oxygen, whereby the electrons were carried from the anode via an external circuit and protons were carried from the anode solution via CEM. There is also a report that the addition of hydrogen peroxide leads to an improvement in power generation [42]. Besides oxygen, there are various electron acceptors [43]; for example, an oxidizing agent such as iron ferricyanide is also used for the cathode. In many cases, the ferricyanide has a high mass transfer efficiency and a high cathode potential so that a high output can be obtained. The combination of carbon electrodes and ferricyanides to achieve power 50–80% higher than the combination of Pt/carbon electrodes and oxygen was reported [44]. In the case of using ferricyanide, once the trivalent iron ion receives the electrons, it becomes divalent, and when it delivers the electrons to oxygen, it reverts to the trivalent state. However, the latter reaction is less likely to occur owing to the low solubility of oxygen. Ferricyanide is an excellent electron acceptor, but owing to its toxicity, its use is generally limited to the laboratory. Other than oxygen and ferricyanide, there are also many candidates, for example, nitrate, persulfate, permanganate, and manganese dioxide. It is also possible to use the nitrate contained in the wastewater because its redox potential is close to that of oxygen, and then, the nitrate is reduced to nitrogen gas at the cathode [43].
